Antifreeze proteins (AFPs) have been identified in many organisms (e.g., fish, plants, and insects) to allow these organisms to survive at sub-freezing temperatures. AFPs adopt diverse structures, but they all noncolligatively depress the freezing point without altering the melting point.
By definition, the equilibrium melting point and freezing points of water are identical. However, thermal hysteresis (TH) proteins, identified in some species of fish, insects, plants, fungi and bacteria have the ability to lower the non-equilibrium freezing point of water without lowering the melting point (equilibrium freezing point). Thus, when TH proteins are added to a solution, they produce a difference between the freezing point and melting temperatures of the solution.
In the absence of a TH protein, a small ice crystal of approximately 0.25 mm in diameter that is about to melt at the melting point temperature will normally grow noticeably if the temperature is lowered by 0.01° to 0.02° C. However, in the presence of a TH protein, the temperature may be lowered by about 1° to 6° C. below the melting point before noticeable ice crystals form, depending upon the specific activity of the protein, the size of the initial ice crystal, the rate of temperature reduction, and the concentration of the protein present. Because of their ability to lower the freezing point of aqueous solutions, these TH proteins are commonly referred to as antifreeze proteins (AFPs). However, unlike antifreezes such as glycerol, antifreeze proteins lower the freezing point of aqueous solutions via a non-colligative mechanism that does not depress the vapor pressure or raise the osmotic pressure of water. Antifreeze proteins also inhibit the recrystallization of ice, a phenomenon wherein grains of small polycrystalline ice change size and shape when held for a prolonged period of time at a sub-melting temperature.
It is generally-accepted that AFPs function through an adsorption-inhibition process. AFPs adsorb onto ice crystal surfaces and produce curved growth fronts of ice. Because of the unfavorable free energy of the curved fronts, the growth of the ice is inhibited. Insect AFPs are usually much more active than fish and plant AFPs. AFPs in fish blood typically produce TH activities of ˜0.7-1.5° C. at ˜20-30 mg/mL and relatively weaker AFPs in plants have TH activities of ˜0.2-0.5° C. AFPs in insect hemolymph (at much lower concentrations compared to those in fish AFPs) are more active, usually having TH activities of ˜3-6° C.
Antifreeze activity of some AFPs can be further enhanced by certain solutes (e.g., sodium chloride, potassium chloride, and glycerol). In the fire-colored beetle Dendroides canadensis, known enhancers of AFPs include some proteins (e.g., antibodies and other AFP isoforms) and common small molecules (e.g., polycarboxylates and polyols). For example, in the Dendroides canadensis hemolymph, glycerol and trehalose have been identified to enhance the antifreeze activity of AFPs from D. canadensis (DAFPs). The purified DAFPs produce less TH activity than the hemolymph itself. These solutes, called enhancers for AFP activity, do not have antifreeze activity themselves.
Noncolligative and natural properties make AFPs superior antifreeze agents over traditional antifreezes, such as glycerol and propylene glycol. At similar concentrations, AFPs are at least 100 times more powerful than common antifreeze agents.
AFPs have been suggested for enhancement of the cryopreservation of biological samples, for inhibition of formation and reformation of clathrate hydrate, for increasing the freezing tolerance of specific fish and plants, and for improvement of the quality of frozen foods. Identification of highly efficient enhancers for antifreeze protein activity can facilitate the commercial application of AFPs. In the presence of enhancers, the antifreeze activity of the AFP solution is enhanced and the amount of AFPs in the solution can be decreased.